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Development of Affordable Bioelectronic Interfaces Using Medically Relevant Soluble Enzymes

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Michigan State University, East Lansing, Michigan. 2006 AIChE Annual Meeting. San Francisco, CA ... Zayats et al., Journal of the American Chemical Society, 124, ... – PowerPoint PPT presentation

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Title: Development of Affordable Bioelectronic Interfaces Using Medically Relevant Soluble Enzymes


1
Development of Affordable Bioelectronic
Interfaces Using Medically Relevant Soluble
Enzymes
2006 AIChE Annual Meeting San Francisco, CA
  • Brian L. Hassler1, Maris Laivenieks2, Claire
    Vieille2, J. Gregory Zeikus2, and Robert M.
    Worden1

1-Department of Chemical Engineering and
Materials Science 2-Department of Biochemistry
and Molecular Biology Michigan State
University, East Lansing, Michigan
2
Presentation Outline
  • Motivation
  • Dehydrogenase enzymes
  • Formation of bioelectronic interfaces
  • Characterization techniques
  • Results
  • Summary

3
Motivation
  • Rapid detection
  • Identification of multiple analytes
  • High throughput screening
  • Affordable fabrication

4
Dehydrogenase Enzymes
  • Catalyze electron transfer reactions
  • Cofactor dependence NAD(P)
  • Challenge cofactor recycling

5
Enzyme Interface Assembly
  • Cysteine branched, trifunctional linker
  • Thiol group self assembles on gold
  • Carboxyl group binds to electron mediator
  • Amine group binds to cofactor
  • Mediator used
  • Toluidine Blue O (TBO)

6
Reaction Mechanism
N-Hydroxysulfosuccinimide
N-(3-Dimethylaminopropyl)-N'-ethylcarbodiimide
Hassler et al., Biosensors and Bioelectronics,
21(11), 2146-2154 (2006)
7
Presentation Outline
  • Motivation
  • Sensing mechanisms
  • Formation of bioelectronic interfaces
  • Characterization techniques
  • Results
  • Summary

8
Chronoamperometry
  • Technique
  • Step change in potential
  • Measure current vs. time
  • Parameters obtained
  • Electron transfer coefficients (ket)
  • Charge (Q)
  • Surface coverage (?)

Zayats et al., Journal of the American Chemical
Society, 124, 14724-15735 (2002)
Katz, E. and I. Willner, Langmuir, 13(13),
3364-3373 (1997)
9
Cyclic Voltammetry
  • Technique
  • Conduct potential sweep
  • Measure current
  • Parameters obtained
  • Sensitivity (slope)
  • Maximum turnover (TRmax)

10
Constant Potential Amperometry
  • Technique
  • Set constant potential
  • Vary analyte concentration
  • Parameters obtained
  • Sensitivity

11
Presentation Outline
  • Motivation
  • Sensing mechanisms
  • Formation of bioelectronic interfaces
  • Characterization techniques
  • Results
  • Summary

12
The Current System
  • Protein array
  • 4 working electrodes
  • Diameter 3 mm
  • Counter electrode
  • Electrode formation
  • Reservoir in PDMS
  • Molecular self-assembly
  • Different enzymes

Polydimethylsiloxane (PDMS)
13
Sorbitol Dehydrogenase (SDH)
  • Organism Pseudomonas sp. KS-E1806
  • Cofactor dependence NAD
  • Temperature stability 30-50?C

14
Chronoamperometric Response
  • Substrate Sorbitol
  • Concentration 5 mM
  • Kinetic parameters
  • k 690 s-1
  • k 87 s-1
  • Surface coverage
  • ? 8.7?10-12 mol cm-2
  • ? 8.0?10-12 mol cm-2

15
Cyclic Voltammetric Response
  • Concentration range 3-21 mM
  • Sensitivity 3.4 mA mM-1 cm-2
  • TRmax38 s-1

16
Amperometric Response
  • Potential -200 mV
  • Concentration range 1-6 mM
  • Sensitivity 2.8 mA mM-1 cm-2

17
Other Enzymes Used
  • Mannitol dehydrogenase
  • Organism Lactobacillus reuteri
  • Reaction Fructose Mannitol
  • Cofactor specificity NAD
  • Thermal stability 50?C-90?C

18
Other Enzymes Used
  • Secondary alcohol dehydrogenase
  • Organism Thermoanaerobacter ethanolicus
  • Reaction 2-Propanol
    Acetone
  • Cofactor specificity NADP
  • Thermal stability 30?C-100?C

19
Chronoamperometric Results
Chronoamperometric measurements were made at a
concentration of 5 mM of the substrate.
20
Cyclic Voltammetry Results
21
Conclusions
  • Developed self-assembling biosensor array
  • Multiple analyte detection
  • Sorbitol
  • Mannitol
  • 2-Propanol
  • Characterized interfaces electrochemically
  • Chronoamperometry
  • Cyclic voltammetry
  • Constant potential amperometry

22
Acknowledgments-
  • Ted Amundsen (CHEMS-MSU)
  • Yue Huang (EECS-MSU)
  • Kikkoman Corporation
  • Funding sources
  • Michigan Technology Tri-Corridor (MTTC)
  • IRGP programs at MSU
  • Department of Education GAANN Fellowship

23
Thank you
  • Questions?

24
Reaction Mechanism
Hassler et. al, Biosensors and Bioelectronics,
77, 4726-4733 (2006)
25
Secondary Alcohol Dehydrogenase (2? ADH)
  • Organism Thermoanaerobacter ethanolicus
  • Cofactor dependence NADP
  • Temperature stability 30-100?C

26
Mannitol Dehydrogenase
  • Organism Lactrobacillus reuteri
  • Cofactor dependence NAD
  • Temperature stability 50-80?C

27
Chronoamperometry response with MDH
  • Electron transfer coefficients
  • ket 5.0102 s-1
  • ket 4.5101 s-1
  • Surface coverage
  • ? 7.210-12 mol cm-2
  • ? 6.010-12 mol cm-2

28
Chronoamperometry response with 2? ADH
  • Electron transfer coefficients
  • ket 6.9102 s-1
  • Surface coverage
  • ? 1.610-11 mol cm-2
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